Continuous cell detection by isotachophoresis

ABSTRACT

A system comprising a protein and a channel. The channel has a domain that binds a membranal component. The channel is configured to carry a liquid sample to an isotachophoresis (ITP) apparatus. The liquid sample comprising or suspected of comprising a cell, a cell membrane or a fraction of a cell membrane. The ITP apparatus comprises a first zone and a second zone. The first zone is configured to contain a solution of high effective mobility leading electrolyte (LE) ion. The second zone is configured to contain a solution of low effective mobility trailing electrolyte (TE) ion. The first zone and the second zone are configured to be operably connected to at least one anode and at least one cathode.

FIELD OF INVENTION

This invention is directed to; inter alia, an isotachophoresis systemfor the detection and/or separation of cells based on a membranalcomponent detectable by a protein.

BACKGROUND OF THE INVENTION

Isotachophoresis

Isotachophoresis (“TTP”) is a variant of electrophoresis, characterizedby the fact that separation is carried out in a discontinuous buffersystem. Sample material to be separated is inserted between a “leadingelectrolyte” and a “terminating electrolyte” or mixed in any of these,the characteristic of these two buffers being that the leader has tohave ions of net mobility higher than those of sample ions, while theterminator must have ions of net mobilities lower than those of sampleions. In such a system, sample components sort themselves according todecreasing mobilities from leader to terminator, in a complex patterngoverned by the so-called Kohlrausch regulating function. The processhas been described repeatedly, as for instance, Bier and Allgyer,Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979).

It is further characteristic of ITP that a steady state is eventuallyreached, where all components migrate at same velocity (hence the name)in sharply defined contiguous zones. Sample components can be separatedin such a contiguous train of components by insertion of “spacers” withmobilities intermediary between those of the components one wishes toseparate.

Isoelectric focusing (“IEF”), also sometimes called electrofocusing, isa powerful variant of electrophoresis. The principle of IEF is based onthe fact that proteins and peptides, as well as most biomaterials, areamphoteric in nature, i.e., are positively charge in acid media andnegatively charged in basic media. At a particular pH value, called theisoelectric point (PI), there is reversal of net charge polarity, thebiomaterials acquiring zero net charge.

If such amphoteric materials are exposed to a d.c. current of properpolarity in a medium exhibiting a pH gradient, they will migrate, i.e.,‘focus’ toward the pH region of their PI, where they become virtuallyimmobilized. Thus a stationary steady state is generated, where allcomponents of the mixture have focused to their respective PIs.

The pH gradient is mostly generated ‘naturally’ i.e, through theelectric current itself. Appropriate buffer systems have been developedfor this purpose, containing amphoteric components which themselvesfocus to their respective PI values, thereby buffering the pH of themedium.

The two variants, IEF and ITP, differ in that IEF attains a stationarysteady state whereas in ITP a migrating steady state is obtained. Thus,in IEF a finite length of migrating channel is always sufficient. InITP, complete resolution may require longer migrating channels than ispractical. In such case, the migrating components can be virtuallyimmobilized by applying a counterflow, the rate of counterflow beingmatched to the rate of frontal migration of the sample ions. This isalso known in the art.

IEF is most frequently carried out in polyacrylamide or agarose gels,where all fluid flow disturbances are minimized. ITP is most oftencarried out in capillaries. The sample is inserted at one end of thecapillary, at the interface between leader and terminator, and themigration of separated components recorded by appropriate sensors at theother end of the capillary. Both such systems are used mainly foranalytical or micro-preparative purposes.

ITP forms a sharp moving boundary between ions of like charge. Thetechnique can be performed with anionic or cationic samples. The systemquickly establishes a strong gradient in electric field at the ITPinterface, due to the non-uniform conductivity profile. As per its name(from Greek, “isos” means “equal”, “takhos” means “speed”), TE and LEions travel at the same, uniform velocity, as a result of thenon-uniform electric field and conservation of current (this is theso-called “ITP condition”).

The ITP interface is self-sharpening: LE ions that diffuse into the TEzone experience a strong restoring flux and return to the leading zone(and vice versa for TE ions in the LE zone). Sample ions focus at thisinterface if their effective mobility in the TE zone is greater thanthose of the TE co-ions, and if their effective mobility in the LE zoneis less than that of the LE co-ions. The self-sharpening and focusingproperties of ITP contribute to the robustness of this technique andmake ITP relatively insensitive to disturbances of the interface (e.g.due to pressure-driven flow or changes in geometry, such ascontractions, expansions, and turns).

In peak mode ITP, sample ion concentrations are at all timessignificantly lower than LE and TE ion concentrations and thereforecontribute negligibly to local conductivity. The distribution of sampleions is determined by the self-sharpening interface between neighboringzones (here the TE and LE) and the value of the sample effectivemobility relative to these zones. Multiple sample ions focus within thesame narrow ITP interface region as largely overlapping peaks.

Pathogen Detection

The conventional bacteria detection methods—e.g. sample cultivation,genotypic detection methods and immunoassays—are time consuming,comprise of several manual steps, and require highly trained personnel.In recent years, there has been significant interest in the use ofmicrofluidic platforms for pathogen detection. Microfluidic technologyenables the manipulation and analysis of small volumes of sample,typically on the order of several nl to several μl and can be leveragedtoward rapid and highly sensitive analysis.

Oukacine et al. (Anal. Chem. 2011, 83, 4949-4954) used simultaneouselectokinetic and hydrodynamic injection with UV detection and thusrequired no labeling. Prior to injection, sample was filtered andisolated from the original water matrix and then resuspended in a lowconductivity electrolyte which was then used in the analysis. Thismethod provided a limit of detection of 2×10⁴ cfu/mL.

Another approach was explored by Phung et al. (Electrophoresis 2013, 34,1657-1662) in order to improve the sensitivity of detection. Their assayinvolves a prelabeling step in which the sample was incubated with SYTO9dye (a cell permeable nucleic acid stain) for approximately 30 min. Thiswas followed by ITP focusing of bacteria from the sample andfluorescence detection of the formed peak. The assay was performed in astandard capillary electrophoresis apparatus and achieved a limit ofdetection of 135 cfu/mL. The authors have also demonstrated thedetection of bacteria at a concentration of ˜10⁴ cfu/mL fromcontaminated river water samples, after filtering the sample to removeparticulates.

However, despite the many advantages of this technology, to date, mostmicrofluidic assays cannot perform continuous analysis and are limitedby their ability to analyze only a single and finite amount of sample,and are thus coupled to significant sample preparation, e.g. byfiltration or centrifugation. This is in contrast to the need forcontinuous and real time monitoring in many pathogen detectionapplications. Thus, there is an unmet need for rapid, continuous,effective, portable and more accurate detection and identification ofinfectious disease-causing pathogens, with the potential of automationand standardization.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a system comprising:

(A) a protein having a domain that binds a membranal component;

(B) a first flow channel configured to provide flow of a liquid sampleto an ITP system, said sample comprising or suspected of comprising acell, a cell membrane or a fraction of a cell membrane; and

(C) an isotachophoresis (ITP) apparatus, said ITP apparatus comprises:

(a) a first zone and a second zone, said first zone is configured tocontain a solution of high effective mobility leading electrolyte (LE)ion, and said second zone is configured to contain a solution of loweffective mobility trailing electrolyte (TE) ion, said first zone andsaid second zone are configured to be operably connected to at least oneanode and at least one cathode; and

(b) at least one second flow channel elongated between said first zoneand second zone;

(D) a flow regulator, said flow regulator is configured to generate aflow countering an electromigration of said protein so as to maintainsaid protein in a pre-determined zone.

In another embodiment, the present invention provides a method fordetecting or sampling a cell of interest, the method comprising thesteps of: (a) providing a labeled protein having a domain that binds amembranal component, wherein said labeled protein is focused by ITP toan ITP zone in a liquid flow channel; (b) applying a counter flow so asto maintain said labeled protein in a stationary zone; (c) providing asample to said flow channel, said sample comprising or suspected ofcomprising a cell of interest, a cell membrane or a fraction of a cellmembrane thereof; and (d) detecting or sampling said cell of interestbound to said labeled protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. (1A) A schematic illustration of an embodiment of the assay.(a) Fluorescently labeled AMPs, initially mixed in the trailingelectrolyte (TE) reservoir, are focused by cationic ITP and heldstationary using vacuum-driven counterflow applied at the TE reservoir.(b) The same vacuum line also continuously draws a flow of potentiallyinfected sample from the leading electrolyte (LE) reservoir. (c) Anybacteria present in the sample travels through the high concentrationAMPs zone, and is instantaneously labeled due to the locally acceleratedreaction. The labeled bacteria continue downstream, while free AMPsremain confined to the stationary ITP zone, thus reducing the downstreambackground signal. (d) Further downstream the fluorescent signal isregistered by a detector. The peaks in the signal, which correspond toindividual bacteria passing through the detector, are counted and yieldthe bacteria concentration in sample. (1B) Fluorescent microscopy imagedemonstrating the labeling of E Coli O:416 with AMPs in the highconcentration zone. A finite sample of AMPs is focused by ITP and heldin place by pressure driven flow opposing the ITP progress. Initiallyunlabeled bacteria that flow through the zone are instantaneouslylabeled and can be clearly seen emerging for the interface. Free AMPsremain contained in the ITP zone.

FIGS. 2A-B. A schematic illustration of an embodiment of themicrofluidic chip layout and experiment setup. (2A) The chip is acommercially available design (NS-12A, PerkinElmer) made ofisotropically etched soda lime glass with dimensions of 90 μm (width)×20μm (depth). Also shown are the length dimensions of each intersectedchannel. A finite amount of AMPs is injected through the West reservoir,focused by cationic ITP, and remains confined and stationary at point(1) by negative pressure applied at the West reservoir. Electric fieldis applied on the channel by setting a constant voltage or currentbetween East and West reservoirs, oriented for cationic ITP propagationfrom the West to the East. Detection of the fluorescent signal isobtained by a camera located at point (2), 4 mm downstream from thelabeling zone. (2B) Raw fluorescence image of the channel intersectionshowing an E. coli sample prelabeled with SYTO9, initially mixed in theSouth reservoir, flowing into the main channel, toward the labelingsite.

FIG. 3. Experimental results demonstrating quantitative bacterialdetection, depicted in a bar graph providing detected bacteria flux(which is defined as the number of detected bacteria per frame) andcorrelated with the original bacterial concentration in sample. Thus,quantitative assessment of the original bacterial concentration wasobtained. No signal was obtained for tap water used as the control case.

FIGS. 4A-C. 4A is a graph depicting experimental measurements ofbacteria counts vs time, demonstrating continuous bacterial detectionfor 1 hour. The obtained signal exhibited stable behavior and increaseslinearly time. The signal was acquired by a CCD camera (iXon, Andor,Belfast Ireland) at The signal was acquired at a 1 Hz frame rate for 1h. Constant voltage of 400 V was applied on the channel. Image analysisfor bacteria detection was performed using MATLAB. 4B shows electriccurrent trace of 1 hour operation of the assay. After an initial stageof formation of labeling zone, the height of the water column ismanually controlled in order to maintain a stable electric current value(approximately 2 μA), indicating that the ITP interface is stationary.4C is a bar graph showing a comparison of bacteria labeling efficiencyfor Escherichia coli (“E. coli”), Bacillus subtilis (“B. subtilis”), andPseudomonas fluorescens (“P. fluorescens”).

FIGS. 5A-B. Experimental results for characterization of labelingefficiency. (5A) Labeling efficiency as a function of applied current.Each measurement corresponds to analysis of pairs of FITC and TRITCimages, acquired in 10 predetermined stations (total set of 20 images).Labeling efficiency was defined as the ratio between the number ofdetected bacteria in the TRITC images and the FITC images. For bacteriaconcentration in the sample, cB, equal to 10⁸ cfu/mL, the number oftotal detected bacteria in 10 stations ranged between 5 and 86 bacteria.The height of each bar represents the average of at least 3 realizations(5 repeats for 2.4 μA; 6 repeats for 6 μA; 3 repeats for 8 μA; 3 repeatsfor 10 μA), with the range of the bars representing 95% confidence ofthe mean. The mean labeling efficiency of the assay is ˜75% regardlessof the applied current value as was supported by t test statisticalanalysis which showed no significant difference (at 95% confidence)between the labeling efficiency at different electric current values.(5B) Dependence of bacteria labeling on AMPs concentration. The resultsare shown as a function of the initial AMPs concentration in thereservoir. Constant current of 3 μA was applied on the channel. Theheight of each bar represents the average of 5 realizations for eachconcentration, with the range of the bars representing 95% confidence ofthe mean. Higher concentration results in better labeling, with nosignificant improvement beyond 0.1 μM as was demonstrated by t teststatistical analysis.

FIGS. 6A-C. Demonstration of the assay's image analysis process. (6A) Arepresentative raw image acquired using a 200 ms exposure, 4 mmdownstream from the ITP interface; (6B) The frame after backgroundcorrection and dilation process; (6C) Final image with boundary traces,after size-based filtering, showing the detected bacteria in the frame.

FIGS. 7A-B. Illustration of the image analysis procedure for measuringlabeling efficiency. (7A) Downstream of the ITP interface zone, 10adjacent fields of view (FOV) are defines along the detection zone. (7B)At each station, two images are acquired at two different wavelengths:using a 480/535 filter for detecting bacteria pre-labeled with SYTO9,and a 545/605 filter for detecting the bacteria labeled on-chip by AMPs.The bacterial detection algorithm is applied to the FITC image and foreach detected bacteria, a search is performed for a corresponding signalin the TRITC image within an 8×8 pixel region around the center of theobject.

FIG. 8. A schematic illustration of an embodiment of the system of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in some embodiments, a micro fluidicsystem and assay for continuous, real-time, and quantitative detectionof pathogens such as bacteria in water.

The present invention is based, in part, on leveraging ITP to focuspeptide probes (e.g., antimicrobial peptides) in a confined region of achannel, through which cells (e.g., bacteria) can flow freely. Thisadvantageously enables continuous labeling, separation, and detection ofa cell (e.g., bacteria) or portions thereof, in a simple microchannel,without requiring any human intervention between steps. As exemplifiedherein below, the assay of the invention provided quantitativemeasurements with a stability of over 1 h of continuous monitoring usingstandard commercially available microfluidic chips.

Thus, in some embodiments the assay disclosed herein enables continuousmonitoring of water at the point-of-need (e.g., water source, watertreatment facilities, municipal networks, and even consumers), relievingthe dependence on clinically trained personnel and eliminating the needto transport samples to a centralized lab. The use of a microfluidicplatform, as well as the significant focusing of labeling probes by ITP,results in a significant reduction in the amount of expensive reagentsrequired for detection and enables online continuous monitoring which isnot possible in other applications. The assay may also be applicable forpathogen detection in food safety and medical diagnostics applications,where rapid pathogen detection is also crucial.

In one embodiment, the present invention provides a system comprising:(A) a protein having a domain that binds a membranal component; (B) afirst flow channel configured to provide continuous flow of a liquidsample to an ITP system, said sample comprising or suspected ofcomprising a cell, a cell membrane or a fraction of a cell membrane; (C)an isotachophoresis (ITP) apparatus comprising a second flow channel;and a (D) a flow regulator, said flow regulator is configured togenerate a flow countering an electromigration of said protein so as tomaintain said protein in a pre-determined zone.

In another embodiment, said ITP apparatus comprises a first zone and asecond zone, said first zone is configured to contain a solution of higheffective mobility leading electrolyte (LE) ion, and said second zone isconfigured to contain a solution of low effective mobility trailingelectrolyte (TE) ion, said first zone and said second zone areconfigured to be operably connected to at least one anode and at leastone cathode; and at least one second flow channel elongated between saidfirst zone and second zone.

In another embodiment, said ITP is an ITP system comprising: (1) a firstzone comprising a solution of high effective mobility leadingelectrolyte (LE) ion; (2) a second zone comprising a solution of loweffective mobility trailing electrolyte (TE) ion; (3) a flow generatingmeans, wherein the flow generating means generates a flow countering anelectromigration of the protein; and (4) an anode and a cathode.

In one embodiment, the present invention provides a system comprising: aprotein having a domain that binds a membranal component; a cell, a cellmembrane or a fraction of a cell membrane; a flow regulator or a flowgenerating means; and an isotachophoresis (ITP) system.

Reference is made to FIG. 8, which illustrates a system 10, constructedand operative in accordance with a non-limiting embodiment of theinvention. System 10 includes a first zone 13 and a second zone 14, suchas that said first zone is configured to contain a solution of higheffective mobility leading electrolyte (LE) ion, and said second zone isconfigured to contain a solution of low effective mobility trailingelectrolyte (TE) ion. For ITP assays, first zone 13 and second zone 14are configured to be operably connected to at least one anode and atleast one cathode. System 10 further includes a flow channel 11 (alsodenoted herein a second flow channel) elongated between said first zoneand second zone. System 10 further includes a flow channel 12 (alsodenoted herein a first flow channel) providing an inlet for sample flow,such as continuous sample flow. In some embodiments, flow channel 12 isconfigured to provide continuous flow of a liquid or aqueous sample toflow channel 11 or first zone 13. System 10 may comprise one or morefilters, such as along flow channel 12, said filters are configured toprovide a sample free of waste which may alter or disturb the ITPfocusing and/or binding of the cell or cell components. It is wellwithin the skill of an ordinary art worker to determine the pore size ofsuch a filter.

Further, system 10 included a flow regulator generate for generating aflow in channel 11, so as to counter an electromigration of a proteinwithin the sample and maintain said protein in a pre-determined zone. Insome embodiments, system 10 further includes a detector 15 as detailedhereinbelow.

In some embodiments, the sample is an environmental sample (e.g.,water). In some embodiments, the sample is an industrial sample and/orfood sample (e.g., liquid foods in raw or processed form, such as milk).Samples may be required to be prepared prior to analysis (e.g.,extraction, dilution, filtration, centrifugation, and/or stabilization).

In another embodiment, said flow generating means is a flow regulator,said flow regulator is configured to generate a flow countering anelectromigration of said protein so as to maintain said protein in apre-determined zone. In another embodiment, the invention provides thatthe flow generating means generates flow countering an electromigrationof the cell (bound or unbound to a protein as described herein). Inanother embodiment, the present invention further provides that the flowgenerating means is electroosmotic or pressure driven.

In another embodiment, a flow regulator or flow generating means isconnected to the liquid tank of the ITP system or the aqueous solutionswithin the ITP. In another embodiment, a flow regulator or flowgenerating means is coupled to the liquid tank of the ITP system or theaqueous solutions within the ITP.

In some embodiments, said binding of a protein having a domain to amembranal component is a semi-selective binding. As used herein“semi-selective” binding refers to binding of the protein to multiplemicrobial species with differing affinities. In some embodiments, saidbinding of a protein having a domain to a membranal component is aselective binding.

In another embodiment, the membranal component is a net charge ofphospholipid head groups. In another embodiment, the membranal componentis a net charge of phospholipid head groups within a defined area of amembrane. In another embodiment, the membranal component is chargedensity of phospholipid head groups. In another embodiment, themembranal component is charge density of phospholipid head groups withina defined area of a membrane.

In another embodiment, the membranal component is a protein. In anotherembodiment, the membranal component is a membranal protein. In anotherembodiment, the membranal component comprises an immunoglobulincomponent. In another embodiment, the membranal component comprises amajor histocompatibility complex (MHC) component. In another embodiment,the membranal component comprises a receptor. In another embodiment, themembranal component comprises a membrane receptor protein. In anotherembodiment, the membranal component comprises a transport protein. Inanother embodiment, the membranal component comprises a membrane enzyme.In another embodiment, the membranal component comprises a cell adhesionmolecule. In another embodiment, the membranal component comprises anintegral membrane protein. In another embodiment, the membranalcomponent comprises an integral polytopic protein. In anotherembodiment, the membranal component comprises a beta barrel protein. Inanother embodiment, the membranal component comprises an integralmonotopic protein. In another embodiment, the membranal componentcomprises a peripheral membrane protein. In another embodiment, themembranal component comprises a toxin. In another embodiment, themembranal component comprises a colicin. In another embodiment, themembranal component comprises a hemolysin. In another embodiment, themembranal component comprises an anti-microbial peptide (AMP)recognition site.

In another embodiment, the cell is an intact cell. In anotherembodiment, “cell” is a eukaryotic cell. In another embodiment, “cell”is a prokaryotic cell. In another embodiment, “cell” is a pathogeniccell. In another embodiment, “cell” is a solitary cell. In anotherembodiment, said cell is within an assembly of cells.

As used herein, the term “pathogen” refers to a microorganism which cancause disease in its host. Examples of pathogens suitable for detectionin accordance with the present disclosure include bacteria, viruses,fungi, prions, and combinations thereof.

None limiting examples of cell that may be detected using the assay andmethod of the invention include: Escherichia coli, Bacillus subtilis,Bacillus cereus, Bacillus thuringiensis, Bacillus coagulans, Bacillusanthracis, Francisella philomiragia, Vibrio cholera, Enterobacteraerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiellaoxytoca, Citrobacter koseri, Citrobacter freundii, Staphylococcusaureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcusfaecium, Streptococcus pneumoniae, Streptococcus pyogenes, Proteusmirabilis, Proteus vulgaris, Serratia marcescens, Morganella morganii,Pseudomonas aeruginosa, Pseudomonas syringae, Stenotrophomonasmaltophilia, Acinetobacter baumannii, Acinetobacter lwoffii,Acinetobacter radioresistens, Acinetobacter johnsonii, Candida albicans,Candida parapsilosis Enterobacteria phage MS2, Influenza A Virus,Influenza B Virus, Cladosporium sphaerospermum, Sacchromyces cerevisiae,and combinations thereof.

In another embodiment, a protein having a domain that binds a membranalcomponent is an antibody. In another embodiment, a protein having adomain that specifically binds a membranal component is a SCFV. Inanother embodiment, a protein having a domain that specifically bin ds amembranal component is an anti-microbial peptide (AMP). In anotherembodiment, a protein having a domain that specifically binds amembranal component is an antibody fragment including single chain,light chain, heavy chain, CDR, E(ab″)2, Fab, Fab′, Fv, sFv, dsFv anddAb, or any combinations thereof. In another embodiment, a proteinhaving a domain that specifically binds a membranal component is amembranal receptor ligand. In another embodiment, a protein having adomain that specifically binds a membranal component is a cell adhesionmolecule binding protein. In another embodiment, a protein having adomain that specifically binds a membranal component is a cell adhesionprotein. Determining and applying a protein having a domain that binds amembranal component to the methods and system described herein is withinthe skill of an ordinary art worker.

In another embodiment, a protein of the invention is a probe. In anotherembodiment, a protein of the invention is labeled. In anotherembodiment, a protein of the invention comprises a fluorescent label. Inanother embodiment, a protein of the invention comprises a radioactivelabel. In another embodiment, a protein of the invention comprises achemiluminescent label. In another embodiment, a protein of theinvention comprises a colorimetric label. In another embodiment, aprotein of the invention serves as a probe and/or as an electricalcharge quencher molecule for the identification and/or separation of amembranal component as described herein.

In another embodiment, the present invention further provides that thesystem as described herein comprises a photodetector, a photomultipliertube (PMT), a conductivity detector, a radioactive detector, a camera orany combination thereof.

In another embodiment, the flow generating means is a pump. In anotherembodiment, the flow generating means is a reciprocating pump. Inanother embodiment, the flow generating means is a rotary pump. Inanother embodiment, the flow generating means is a mechanical pump. Inanother embodiment, the flow generating means is any pump known to oneof skill in the art. In another embodiment, the flow generating means orpump generates a continuous flow. In another embodiment, the flowgenerating means or pump generates a uniform outflow. In anotherembodiment, the flow generating means or pump generates a uniformpressure. In another embodiment, the flow generating means or pump canbe adjusted in terms of its pumping capacity, its outflow generation,its pressure generation or any combination thereof. In anotherembodiment, the flow generating means is adjusted to equally counter theflow of the protein of the invention. In another embodiment, the flowgenerating means is responsible for maintaining the stationary portion(non-migrating zone for the unbound protein) of the ITP wherein freeprotein is present. In another embodiment the sum of ITPelectro-migration and counter-flow generated by the flow generatingmeans with respect to unbound protein within the ITP system as describedherein, is zero. In another embodiment, flow countering anelectromigration is electroosmotic or pressure driven.

In another embodiment, the stationary zone is an anionic stationary zonecharacterized by high concentration of the protein. In anotherembodiment, the stationary zone is an anionic stationary zonecharacterized by high concentration of unbound protein. In anotherembodiment, the stationary zone is an anionic stationary zone free orsubstantially free of bound protein or protein complexes. In anotherembodiment, the stationary zone is an anionic stationary zone comprisingall or most of the unbound protein with the ITP system.

In another embodiment, the stationary zone is a cationic stationary zonecharacterized by high concentration of the protein. In anotherembodiment, the stationary zone is a cationic stationary zonecharacterized by high concentration of unbound protein. In anotherembodiment, the stationary zone is a cationic stationary zone free orsubstantially free of bound protein or protein complexes. In anotherembodiment, the stationary zone is a cationic stationary zone comprisingall or most of the unbound protein with the ITP system.

In another embodiment, the leading electrolyte (LE) buffer is chosensuch that its ions (cations or anions) have higher effectiveelectrophoretic mobility than the ions of the trailing electrolyte (TE)buffer. Effective mobility describes the observable drift velocity of anion and takes into account the ionization state of the ion, as describedin detail by Persat et al. In another embodiment, sample ions ofintermediate effective mobility race ahead of TE ions but cannotovertake LE ions, and so they focus at the LE-TE interface (hereinaftercalled the “ITP interface”). In another embodiment, the LE and TEbuffers are chosen such that have a higher mobility than the TE, butcannot overspeed the LE. In another embodiment, the TE and LE buffersform regions of respectively low and high conductivity, which establisha steep electric field gradient at the ITP interface. In anotherembodiment, the LE buffer (or LE) has a high ionic strength. In anotherembodiment, the LE buffer comprises Sodium hydroxide. In anotherembodiment, Mg²⁺ ions are used as a counter ion to promote rapidhybridization. In another embodiment, TE buffer (or TE) comprisesPyridine. In another embodiment, LE comprises hydrochloric acid. Inanother embodiment, LE comprises 70 to 150 mM HCl and 150 to 280 mMBistris (2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol).

In another embodiment, LE comprises NaOH. In another embodiment, LEcomprises 70 to 100mM NaOH. In another embodiment, LE comprises NaOH. Inanother embodiment, LE comprises 100 to 150 mM NaOH. In anotherembodiment, LE comprises NaOH. In another embodiment, LE comprises 120to 150mM NaOH. In another embodiment, LE comprises 150 to 200 mM Hepes.In another embodiment, LE comprises 200 to 250 mM Hepes. In anotherembodiment, LE comprises 150 to 200 mM Bistris. In another embodiment,LE comprises 220 to 280 mM Hepes.

In another embodiment, ITP includes a microchannel connected to tworeservoirs and is initially filled with LE solution. In anotherembodiment, a sample comprising a cell to be detected is mixed in thetrailing electrolyte (TE) reservoir. In another embodiment, a samplecomprising cell to be detected is mixed in the leading electrolyte (LE)reservoir. In another embodiment, an electric field induces theelectromigration of all ions in the channel.

In another embodiment, a system as described herein further comprises aphotodetector. In another embodiment, a system as described hereinfurther comprises a photomultiplier tube (PMT). In another embodiment, asystem as described herein further comprises a camera. In anotherembodiment, a system as described herein further comprises a radioactiveprobe or detector. In another embodiment, a system as described hereinfurther comprises a calorimetric detector. In another embodiment, asystem as described herein further comprises a conductivity detector.

In another embodiment, the present invention further provides a methodfor detecting a cell of interest, comprising the steps of: (a) Settingthe ITP system of claim 1, wherein said protein is a labeled protein;(b) Applying an electric field across the first zone and the secondzone; (c) Calibrating said flow generating means to create: (1) astationary zone characterized by a high concentration of the labeledprotein, and (2) a continuous flow of the cell of interest from thefirst zone to the second zone; (d) Adding a sample comprising the cellof interest through said flow generating means; and (e) Detecting thecell of interest bound to the labeled protein; Wherein both the cell ofinterest bound to the labeled protein and unbound cell of interest flowfrom the first zone to the second zone while unbound labeled proteinremains within the stationary zone, thereby detecting a cell ofinterest.

In another embodiment, the present invention provides a method fordetecting a cell of interest, the method comprising the steps of: (a)providing a labeled protein having a domain that binds a membranalcomponent, wherein said labeled protein is focused by ITP to an ITP zonein a liquid flow channel; (b) applying a counter flow so as to maintainsaid labeled protein in a stationary zone; (c) providing a sample tosaid flow channel, said sample comprising or suspected of comprising acell of interest, a cell membrane or a fraction of a cell membranethereof; and (d) detecting said cell of interest bound to said labeledprotein.

In another embodiment, the present invention provides a method fordetecting and/or sorting cells. In another embodiment, the presentinvention provides a method for detecting and/or sorting cells whereincells are continuously introduces into one of the aqueous solutions (TEor LE). In another embodiment, the present invention provides a methodfor detecting and/or sorting cells wherein cells are continuouslyinfused into one of the aqueous solutions (TE or LE). In anotherembodiment, the present invention provides that the cells arecontinuously infused via the flow generating means. In anotherembodiment, the present invention provides that the cells arecontinuously infused via an infusing means.

In another embodiment, the term “detecting” includes labeling,separating, enriching, identifying, sorting, isolating, or anycombination thereof. In another embodiment, detecting is quantitative,qualitative, or both. In another embodiment, detecting is achieved byusing a photodetector, a sensor, an affinity column, a photomultipliertube (PMT), a conductivity detector, a radioactive detector, a lightdetector, an emission detector, a camera or any combination thereof.

In another embodiment, the present invention provides an ITP kitcomprising the system as described herein and specific instructions forperforming the method as described herein. In another embodiment, thepresent invention provides a kit comprising an instruction manualdescribing the method and/or system disclosed herein. In anotherembodiment, the present invention provides a kit as described hereinfurther comprising an electrophoresis apparatus. In another embodiment,the present invention provides a kit as described herein furthercomprising an electrophoresis apparatus that is communicatively coupledto a central processing unit (including but not limited to a centralprocessing unit (CPU), ASIC or FPGA, that may operate theelectrophoresis apparatus based on a predetermined set of instructions.

In another embodiment, detecting is detecting a specific cell type or afragment thereof. In another embodiment, detecting is detecting cellmarker on the cell's membrane. None limiting examples of pathogens thatcan be detected in water samples using the methods described hereininclude protozoa such as those of the genus Cryptosporidium and thegenus Giardia; bacteria such as Escherichia coli, Yersinia pestis,Francisella tularensis, Brucella species, Clostridium perfringens,Burkholderia mallei, Burkholderia pseudomallei, Chlamydia psittaci,Coxiella burnetii, Rickettsia prowazekii, Vibrio species; Enterococcusfaecalis; Staphylococcus epidermidis; Staphylococcus aureus;Enterobacter aerogenes; Corynebacterium diphtheriae; Pseudomonasaeruginosa; Acinetobacter calcoaceticus; Klebsiella pneumoniae;Serratia; yeasts such as Candida albicans; and viruses, includingfiloviruses such as Ebola and Marburg viruses, naviruses such as Lassafever and Machupo viruses, alphaviruses such as Venezuelan equineencephalitis, eastern equine encephalitis, and western equineencephalitis, rotoviruses, calciviruses such as Norwalk virus, andhepatitis (A, B, and C) viruses, and biological warfare agents such assmallpox (i.e., variola major virus).

In another embodiment, the system of the invention is set includingintroducing the protein that may serve as a probe into the TE or LEsolution, applying electrical field between the LE and the TE zones anda counterflow at the same time while adjusting the electrical field andthe counterflow to values that permit the formation of a stationaryzone-wherein the protein is captured, actuating the detection means, andadding a cell or a cell fragment, wherein cells or cell fragmentslabeled by the protein but not unbound protein-migrate and wherein adetector set at a location after the stationary zone in the direction ofelectromigration is configured to detect protein labeled membranes butnot cells free of the protein probe. In another embodiment, a detectorset at a location after the stationary zone in the direction ofelectromigration is configured to detect and differentiate between bothprotein labeled membranes and cells free of the protein probe. Inanother embodiment, a detector set at a location after the stationaryzone in the direction of the flow is configured to separate betweenprotein labeled membranes and cells free of the protein probe.

In another embodiment, a method, a system and a kit as described hereinmay include a separation and/or isolation in one device or two separatedevices. In another embodiment, the second step includes subjecting themigrating protein labeled cells to an electric field thus furtherisolating these probed cells according to their isoelectric point. Inanother embodiment, the separating means is a column which is capable ofseparating/distinguishing “naked” cells from protein probed cells.

In another embodiment, the present invention provides methods, systemsand kits that reduce false positive or false negative results. Inanother embodiment, the present invention provides methods, systems andkits that reduce background noise. In another embodiment, the presentinvention provides methods, systems and kits that provide accuratequantitative measurements of cells of interest. In another embodiment,the present invention provides methods, systems and kits that provide anefficient separating technique for a cell of interest. In anotherembodiment, the present invention provides methods wherein the free,unhhybridized, protein is focused in a stationary zone.

In another embodiment, the protein is labeled. In another embodiment,the label is Acridine orange. In another embodiment, the label isAcridine yellow. In another embodiment, the label is Alexa Fluor. Inanother embodiment, the label is 7-Aminoactinomycin D. In anotherembodiment, the label is 8-Anilinonaphthalene-1-sulfonic acid. Inanother embodiment, the label is an ATTO dye. In another embodiment, thelabel is Auramine-rhodamine stain. In another embodiment, the label isBenzanthrone. In another embodiment, the label is Bimane. In anotherembodiment, the label is 9,10-Bis(phenylethynyl)anthracene. In anotherembodiment, the label is 5,12-Bis(phenylethynyl)naphthacene. In anotherembodiment, the label is Bisbenzimide. In another embodiment, the labelis a Blacklight paint. In another embodiment, the label is Brainbow. Inanother embodiment, the label is Calcein. In another embodiment, thelabel is Carboxyfluorescein. In another embodiment, the label isCarboxyfluorescein diacetate succinimidyl ester. In another embodiment,the label is Carboxyfluorescein succinimidyl ester. In anotherembodiment, the label is 1-Chloro-9,10-bis(phenylethynyl)anthracene. Inanother embodiment, the label is2-Chloro-9,10-bis(phenylethynyl)anthracene. In another embodiment, thelabel is 2-Chloro-9,10-diphenylanthracene. In another embodiment, thelabel is Coumarin. In another embodiment, the label is DAPI. In anotherembodiment, the label is a Dark quencher. In another embodiment, thelabel is DiOC6. In another embodiment, the label is DyLight Fluor. Inanother embodiment, the label is Ethidium bromide. In anotherembodiment, the label is Fluo-3. In another embodiment, the label isFluo-4. In another embodiment, the label is a FluoProbe. In anotherembodiment, the label is Fluorescein. In another embodiment, the labelis Fluorescein isothiocyanate. In another embodiment, the label is aFluoro-Jade stain. In another embodiment, the label is Fura-2. Inanother embodiment, the label is Fura-2-acetoxymethyl ester. In anotherembodiment, the label is GelGreen. In another embodiment, the label isGelRed. In another embodiment, the label is Green fluorescent protein.In another embodiment, the label is a Heptamethine dye. In anotherembodiment, the label is Hoechst stain. In another embodiment, the labelis Indian yellow. In another embodiment, the label is Indo-1. In anotherembodiment, the label is Lucifer yellow. In another embodiment, thelabel is Luciferin. In another embodiment, the label is MCherry. Inanother embodiment, the label is Merocyanine. In another embodiment, thelabel is Nile blue. In another embodiment, the label is Nile red. Inanother embodiment, the label is an Optical brightener. In anotherembodiment, the label is Perylene. In another embodiment, the label isPhloxine. In another embodiment, the label is P cont. In anotherembodiment, the label is Phycobilin. In another embodiment, the label isPhycoerythrin. In another embodiment, the label is Phycoerythrobilin. Inanother embodiment, the label is Propidium iodide. In anotherembodiment, the label is Pyranine. In another embodiment, the label is aRhodamine. In another embodiment, the label is RiboGreen. In anotherembodiment, the label is RoGFP. In another embodiment, the label isRubrene. In another embodiment, the label is (E)-Stilbene. In anotherembodiment, the label is (Z)-Stilbene. In another embodiment, the labelis a Sulforhodamine. In another embodiment, the label is SYBR Green I.In another embodiment, the label is Synapto-pHluorin. In anotherembodiment, the label is Tetraphenyl butadiene. In another embodiment,the label is Tetrasodium tris(bathophenanthrolinedisulfonate)ruthenium(II). In another embodiment, the label is TexasRed. In another embodiment, the label is Titan yellow. In anotherembodiment, the label is TSQ. In another embodiment, the label isUmbelliferone. In another embodiment, the label is Yellow fluorescentprotein. In another embodiment, the label is YOYO-1. In anotherembodiment, the label is a chemiluminescent dye. In another embodiment,the label is a radioisotope or a radioactive dye. In another embodiment,the label is a dye that can be detected by a naked eye.

In another embodiment, the protein is a peptide. In another embodiment,the protein is a polypeptide. In another embodiment, the protein is aglycoprotein.

In another embodiment, the invention provides that cells migrate whereinunbound protein remains within the stationary zone. In anotherembodiment, the invention provides that protein probed cells migrateslower compared to unprobed cells.

In another embodiment, the method of the present invention can beutilized to identify pathogens. In another embodiment, the method of thepresent invention can be utilized to identify certain bacteria. Inanother embodiment, the method of the present invention can be utilizedto identify any bacteria. In another embodiment, the method of thepresent invention can be utilized to identify a desired cell type suchas but limited to a transfected cell, an immune cell, a cancer cell, ablood cell, a muscle cell, or any other known cell type carrying anidentifiable mark on its membrane.

In another embodiment, the present method requires minimal or no samplepreparation. In another embodiment, the theory behind ITP is provided inBahga S S, Kaigala G V, Bercovici M, Santiago J G. High-sensitivitydetection using isotachophoresis with variable cross-section geometry.Electrophoresis. 2011 February; 32(5):563-72; Khurana T K, Santiago J G.Sample zone dynamics in peak mode isotachophoresis. Anal Chem. 2008 Aug.15; 80(16): 6300-7; and Isotachophoresis: Theory, Instrumentation andApplications. F. M. Everacrts, J. L. Beckers, T.P.E.M. Verheggen,Elsevier, Sep. 22, 2011, which are hereby incorporated by reference intheir entirety.

In another embodiment, ITP is performed in a peak mode. In anotherembodiment, ITP is performed in a plateau mode. In another embodiment,“Plateau mode” refers to a wide sample-zone compared to the transitionzones, i.e. the sample concentration distribution forms a plateau withblurred boundaries towards LE and TE. In another embodiment, “Peak mode”refers to a very short sample zone, where the two transition zones atboth sides of the sample overlap or when the sample is entirely withinthe interface between LE and TE.

The sensitivity of the assay described herein is governed by theincoming flow rate of the sample. Thus, in some embodiments, largerchannel dimensions can be used for achieving improved sensitivity. Themicrofluidic channel exemplified herein is a standard commerciallyavailable design (90 μm wide and 20 μm deep) and provided a LOD ofapproximately 10⁴ cfu/mL over a 60 min detection window. A set of 100such parallel channels would have a total width of only 1 cm and wouldenable a LOD of approximately 10² cfu/mL over a 1 h window.

In additional embodiments, increased flow rate can be achieved bymaximizing the applied electric field on the channel, thereby increasingflow velocity. The labeling efficiency experiments demonstratedhereinbelow revealed that at least a 10-fold increase in the appliedcurrent is possible without affecting the labeling efficiency andimpairing detection. Hence, designing a dedicated microfluidic chip,using the same principles presented here, may allow one to detect aconcentration as low as 10² cfu/mL in minutes.

In additional embodiments, higher throughput could also be achieved byusing a larger diameter channel or capillary, but as temperature due tojoule heat scales with the diameter, this would lead to excessiveheating and require specialized cooling. In contrast, the use ofmultiple parallel channels in a planar format maintains the depth of thechannel, and thus, temperature is expected to remain essentiallyunchanged. Higher throughput and longer analysis time would also requirescaling the size of the reservoirs to avoid pH changes due tohydrolysis. As detailed by Persat et al. (Chips & Tips (Lab on a Chip),2007; 1-8), operating the assay at 200 μA with a 100 mM LE, withoutexceeding a pH change of 0.2 in the reservoirs, would require areservoir volume of 1.2 mL for 10 h of operation. This is a sufficientlysmall volume to be easily integrated with a microfluidic system.

Reference is made to Examples 1-2, demonstrating the assay of theinvention using AMPs, which are relevant for detecting bacteria but donot provide information on the bacterial strain or species. Nonspecificdetection of bacteria is useful in water monitoring as an early alertstep and a first warning sign but still cannot be compared to theadvantages of specific detection. In some embodiments, specificantibodies may be used as probes for particular pathogen strains. Asmany bacteria species are negatively charged over a wide range of pH(Harden and Harris, Bacteriol. 1953, 65, 198), it may be necessary toapply conditions in which the antibodies are positively charged andremain active, and their effective mobility is bracketed between thoseof the leading and trailing electrolytes. Most antibodies produced inmammals have pI values between 6.1 and 8.5 (Amersham Pharmacia BiotechAntibody Purification: Handbook; Amersham Pharmacia Biotech: Piscataway,N.J., 2000). Thus, by designing a cationic ITP buffer system at a pH <6,it may be possible to focus the majority of antibodies and apply theassay disclosed herein for detection of specific bacteria strains orspecies.

The method demonstrated here also has an inherent capability to operatewith the use of only very small volumes of reagents due to the ITPfocusing. The typical reagent amounts used in other AMP-based methodsfor bacteria detection ranges between 450 and 2500 ng. In contrast, theassay disclosed herein may use only 4.6 ng of AMPs in the reservoir.This amount can be even further reduced by allowing more focusing of theAMPs at the ITP interface during the formation of the labeling zone.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Strategies for ProteinPurification and Characterization—A Laboratory Course Manual” CSHL Press(1996); all of which are incorporated by reference. Other generalreferences are provided throughout this document.

Materials and Methods

Chemicals and AMPs.

ITP Buffers. Cationic ITP was performed using LE buffer composed of 100mM NaOH and 200 mM HEPES and TE buffer composed of 10 mM pyridine and 20mM HEPES. To suppress electroosmotic flow (EOF), ˜1% 1 MDapoly(vinylpyrrolidone) (PVP) was added to both buffers. All buffercomponents were purchased from Sigma-Aldrich, (St. Louis, Mo.) andprepared in purified UltraPure DNase/RNase free distilled water (Milli-Qwater purification, Millipore Corp., Billerica, Mass., USA).

AMPs. TAMRA labeled Indolicidin, 5-TAMRA-ILPWKWPWWPWRR (SEQ ID NO: [1]),synthesized by Biomatik (Wilmington, Del., USA) was used in the currentstudy. Indolicidin is present in nature in bovine neutrophils anddemonstrates broad spectrum of activity against Gram-negative andGram-positive bacteria, fungi, and protozoa. A stock solution wasprepared by solubilizing the AMPs in 1 to 8 acetonitrile in deionizedwater and stored at −20° C. Further dilutions were freshly prepared inacetonitrile before each set of experiments from a stock solutionconcentration of 100 μM which was kept refrigerated.

The peptide properties were estimated using the PepCalc.com onlinecalculator. The pI of the peptide is at pH 12.41, and at a neutral pH,the overall charge is +2. Thus, it can likely be considered as a fullyionized cationic species in all experiments.

The AMPs mobility was estimated to be around 5×10⁻⁹ m²/(Vs) by observingthe focusing of sample in a set of ITP experiments with various TEcompositions having different effective mobilities. A tendency of theAMPs to attract and adhere to the negatively charged channel walls wasnoticed, likely due to electrostatic interaction. Thus, stringentcleaning of the channels between experiments was necessary. In addition,at very high concentrations (above 10 μM), aggregation and precipitationof AMPs was noticed, which can result in precipitants flowing toward thedetector causing false positives. To avoid this, smaller concentrationsof AMPs were used (as indicated hereinbelow) and introduced to thechannel only a finite amount of AMPs which remains at a solubleconcentration when focused.

Bacteria Growth Conditions and Sample Preparation.

Bacteria Growth Conditions. E. coli O:416 was used as a model strain inthis study. Stock cultures were prepared by incubating E. coli in Luriabroth (LB) at 37° C. to an OD600 of 0.3, corresponding to approximately3×10⁸ cfu/mL, as measured by standard plating. Thereafter, 1 mL of cellsuspensions was transferred to sterilized 1.5 mL vials, centrifuged at14650 rpm for 2 min, and discarded the supernatant. The centrifugationprocess was repeated twice to remove any medium remnants and stored thebacteria pellets at −20° C.

Sample Preparation. For experiments performed with unlabeled bacteria,pellets were resuspended in 1 mL of tap water and serially diluted intap water to the final concentrations. For labeling efficiencyexperiments performed with prelabeled bacteria, the pellets wereincubated in 200 μL of tap water and 4 μL of 34 μM SYTO9 BacLight(LIVE/DEAD BacLight Bacterial Viability Kit, Life technologies) for 10min. To discard remaining free fluorophores, the pellet was centrifugedat 14650 rpm for 2 min, then removed supernatant, and resuspended thepellets in 1 mL of tap water, for a final concentration of 3×10⁸ cfu/mL.

Experimental and Imaging Settings.

All experiments were performed on a NS-12A microchip made ofisotropically etched soda lime glass (PerkinElmer, Waltham, Mass., USA).An overview of the chip geometry is provided in FIG. 2. The chipconsists of two intersecting channels, each having maximum width anddepth of 90 and 20 μm, respectively. Images were obtained using aninverted epifluorescent microscope (Ti-U, Nikon, Tokyo, Japan) equippedwith a metal halide light source (Intensilight, Nikon Japan), 20×objective (Plan Fluor, NA=0.75, WD=0.66 mm, Nikon, Tokyo, Japan), TAMRAcompatible filter-cube (model 49004, 545/25 nm excitation, 605/70 nmemission, and 565 nm dichroic mirror, Chroma, Bellows Falls, Vt., USA),SYTO9 compatible filter-cube (480/15 nm excitation, 535/20 nm emission,and 505 nm dichroic mirror, Nikon, Tokyo, Japan), and motorized stage(MS-2000, Applied Scientific Instrumentation, Eugene, Oreg.). Imageswere captured using a 14 bit, 512×512 pixels CCD camera (Clara, Andor,Belfast, Ireland). The camera and stage were controlled using NISElements software (v.4.11, Nikon, Tokyo, Japan) and processed the imageswith MATLAB (R2011b, Mathworks, Natick, Mass.). Constant voltage orcurrent were applied using a sourcemeter (model 2410, KeithleyInstruments, Cleveland, Ohio).

Assay Procedure.

Before each experiment, the channel was cleaned by sequentially flowingbleach and DI water, for 2 min each. In each experiment, first, astationary ITP zone containing focused AMPs was established and then thesample was introduced into the channel.

Initial Formation of Labeling Zone. For chip loading, the North, East,and South reservoirs were filled with 20 μL of LE and applied vacuum atthe West reservoir for 1 min to fill the channel. Next, the Westreservoir was rinsed with DI water and filled it with 18 μL of TE and 2μL of 1 μM AMPs. Constant voltage was applied between the West and Eastreservoirs. When the focused AMPs plug traveled a distance of 6 mm alongthe channel, the voltage was stopped, the West reservoir was rinsed, and20 μL of pure TE was filled in it. Thereafter, the voltage (or current)was reapplied to regain focusing of the injected AMPs. This resulted ina finite and well controlled amount of focused AMPs. To then hold theAMPs plug stationary, negative pressure was applied to the Westreservoir using a water column, resulting in pressure driven flowcountering electromigration. The flow rate was controlled by changingthe height of the water column according to visual monitoring of the ITPinterface progression and feedback on the current reading, which is agood indication for the location of the interface in the channel. TheAMPs plug, i.e., the labeling reaction zone, was positioned stationary12 mm from the West reservoir (marked as station (1) in FIG. 2).

Detection Procedure. 10 μL of the sample of interest was introduced tothe South reservoir followed by actuation of the pipet several times tohomogenize the solution. For time dependent detection, the objective waspositioned 4 mm downstream from the labeling reaction zone (marked asstation (2) in FIG. 2) and the camera was triggered at 5 Hz for 2 min.For labeling efficiency experiments, the same procedure was used, butthe spiked samples were also prelabeled with SYTO9 (labeling proceduredescribed above). After turning off the electric field (and counterflow), images were captured at 10 stations downstream from the interfacezone using both FITC and TRITC filters (total set of 20 images).

Bacteria count. In the continuous mode of the assay, the signalcorresponds to a bacterial count in each recorded frame (ultimatelyproviding the bacterial flux). As illustrated in FIG. 6, each raw imageis analyzed using the following procedure implemented in Matlab: Sincebacteria are sparse elements in each frame, the average intensity valueof the frame is treated as an approximation for the background noise.The background is subtracted from the entire image. Further, ahorizontal 4 pixels length line structuring element is constructed,which is used to dilate the background subtracted image. Boundary andedge trace functions are then used to find the boundaries of allelements in the dilated image and are finally, discarded by size andangle conditions the “noise” elements, and remaining closed regions arecounted as bacteria.

Labeling efficiency. As illustrated in FIGS. 7A-B, pairs of FITC andTRITC images, acquired at 10 predetermined stations, are analyzed. Thesame bacteria count procedure as described herein was used to traceseparately the bacteria in the FITC and TRITC images. For each bacteriafound in the FITC image (corresponding to the SYTO9 channel), a regionof 8×8 pixels in the corresponding TRITC image was searched for anoverlapping signal. The labeling efficiency was calculated as the rationbetween the total number of elements detected in the TRITC images andthe number of element detected in the FITC image.

Current monitoring. A constant voltage of 400V was applied on thechannel. Initially, there is a decrease in current which indicates theformation of a propagating ITP front in the channel. At approximately 40sec, the voltage is switched off as part of the AMPs injection process(TE reservoir is cleaned and replaced with pure TE), and thenreestablished to regain focusing. Negative pressure is applied bychanging the height of the water column connected to the TE reservoir.This results in pressure driven flow that opposes the ITP interfacemigration, and maintains in stationary. As the interface stabilizes inplace, so does the electric current value. Tracking the current changein real time, the high tog the water column is manually adjusted to keepthe current stable and thus hold the ITP zone at the same location.

Example 1 Microfluidic Assay for Continuous Real-Time Pathogen DetectionUsing Antimicrobial Peptides and Isotachophoresis

The basic assumption for the following experiment was that PNAstargeting bacteria by non-specific binding to their negatively chargedouter-membrane can be used as the protein as described herein and thebacteria to be detected can be the cell to be detected as describedherein.

An ITP system such as illustrated in FIG. 1 was used for the currentexperiments. The system included the settings as described herein.Specifically, positively charged labeled AMPs are universal probes forlabeling and detecting bacteria. Using cationic ITP focusing, a highconcentration zone of AMPs within a microchannel was formed. A pressuredriven flow countering electromigration was applied to hold the zonestationary. Through this “virtual reaction chamber” the sample ofinterest was flowing (from the LE reservoir). Any bacteria present inthe sample was simultaneously labeled by the AMP, and separated from,the high concentration AMPs, and continued downstream to a detector.This enabled continuous, real-time, quantitative one step labeling anddetection of bacteria in sample. This experiment included ITP withfluorescently-labeled anti-microbial peptides (AMPs).

FIGS. 3 and 4 demonstrate the ability of the method to achievecontinuous and quantitative bacteria detection in water samples. FIG. 3presents the measured bacteria flux versus the known bacteriaconcentration introduced in the reservoir. Here, the bacteria in thesample arc originally unlabeled and obtain their fluorescent labeling asthey pass through the high concentration AMPs region. Images arerecorded at a distance of 4 mm downstream from the ITP interface, andthe number of bacteria in each frame is counted, as describedhereinbelow. After binding to the outermembrane of the target bacteria,the AMPs may lyse and disrupt it as part of their killing mechanism.However, as this process typically takes several minutes and thedetection of the assay described herein takes place only a few secondsafter the binding, significant effect on the detection is not expected.

The acquired signal (i.e., bacterial flux) is proportional to theoriginal bacterial concentration in the sample, illustrating thatquantitative detection of E. coli can be obtained. Two sample t testmethod were used to determine whether the difference between any twomeasured mean values is significant. A p-value of p=0.05 (correspondingto 95% confidence on a statistical difference between results) wasdefined. In all cases, the calculated t-values were significantly higherthan the required threshold (for example, the calculated t-value for thedifference between the signal at 10⁶ and 10⁷ cfu/mL is 5.26, whereas thethreshold based on the number of repeats is 2.57), indicating clearstatistical significance. The limit of detection is determined by theflow rate of sample into the channel. Here, for channel cross section of90 μm×20 μm, ITP velocity of u_(ITP)=100 μm/s, and approximated bacteriamobility of μ_(B)=−30×10⁻⁹ m²/(V s), the bacterial flux for the 10⁸cfu/mL sample is 1542 cfu/min. This is in good agreement with the orderof magnitude of measured experimental values. Under these conditions,the lowest concentration detected in 2 min was 10⁶ cfu/mL; in this timeperiod, approximately 40 bacteria pass through the ITP interface, getsufficiency labeled, and are detected. The extrapolated limit ofdetection is thus 10⁵ cfu/mL (yielding 4 bacteria in 2 min).

Using the same cross-section geometry, better limits of detection couldbe obtained with longer monitoring times. FIG. 4A presents bacteriacount versus time and demonstrates continuous operation of the assay forover an hour. At a concentration of 10⁸ cfu/mL, we obtain approximately9000 detections per hour (or 150 per minute). Importantly, the number ofdetected bacteria grows at a constant rate, substantiating andvalidating the assay stability and possible use as a continuous watermonitoring platform. To further support the claim of the assaystability, a trace of the monitored current during the whole procedureis shown in FIG. 4B. Furthermore, no bacteria aggregation or channelclogging was observed during this 1 h time frame. As measured by Hardenand Harris (Bacteriol. 1953, 65, 198) many other bacterial species arealso negatively charged over a wide range of pH values, enabling theirdetection using the present assay. FIG. 4C presents the applicability ofthe assay to other (Gram-negative and Gram-positive) bacteria species.

The signal was acquired at 5 Hz frame rate for 2 min. The height of eachbar represents the average of at least 5 realizations, with the range ofthe bars representing 95% confidence of the mean. Constant voltage of400 V was applied on the channel, resulting in a current ofapproximately 2 μA.

Experimental measurements of bacteria counts vs time, demonstratingcontinuous bacterial detection during a 1 h period. The obtained signalexhibits stable behavior and increases linearly with time, suggesting nosignificant deterioration of the finite AMPs sample focused at theinterface. The control sample contained only tap water and shows only amoderate increase, after long times, likely due to autofluorescence ofcontaminants or precipataion of AMPs. The signal was acquired at a 1 Hzframe rate for 1 h. Constant voltage of 400 V was applied on thechannel.

Thus the present experiment enabled:

-   -   1. The design and use of positively charged labeled AMPs as        universal probes for labeling and detecting bacteria.    -   2. The design of an ITP system (with an adequate chemistry)        under which the AMPs are focused.    -   3. The formation of a high concentration AMPs zone that serves        as a “virtual reaction chamber” for accelerated rapid binding of        AMPs to bacteria outer membrane, thus labeling the bacteria.    -   4. Ability to continuously label, separate and detect bacteria        in free solution, in one step.    -   5. Ability to quantify the initial bacteria concentration in        sample, using the same method.

Example 2 Labeling Efficiency

The sensitivity of the assay is directly affected by the flow rate, aswell as the labeling efficiency of bacteria as it passes through theAMPs confined at the LE-TE interface. To characterize the latter, theassay was performed on tap water samples spiked with bacteria which wereprelabeled with SYTO9. After stabilizing the assay, the counterflow andvoltage were simultaneously stopped, such that all bacteria remainstationary. The channel was imaged at 10 stations downstream of the ITPinterface zone. At each station, images were taken using two filters:480/535 for detecting the bacteria prelabeled with SYTO9 and 545/605 fordetecting the bacteria which were labeled on-chip by AMPs. SYTO9 emitsat a wavelength of 500 nm, which is sufficiently shifted from the 579 nmemission of the TAMRA labeled AMPs, and thus, it is possible to measurethe number of bacteria which were successfully labeled and compare itwith the total number of bacteria that passed through the labeling zone.It should be clarified that the use of SYTO9 dye was not to monitor theviability of the bacteria but only to be able to count the total numberof bacteria in frame. The labeling efficiency was defined as the ratiobetween the number of detected AMPs labeled bacteria and the totalbacteria present in all the frames. To obtain additional statistics, theprocess was repeated several times by reapplying the current andcounterflow to fill the channel with a new set of labeled bacteria.Imaging after stopping the flow enables one to perform thiscolocalization test of the signal, which is not possible during thestandard assay operation. FIG. 7 illustrates this detection process.

The influence of both the applied current (affecting theelectromigration speed of the bacteria) and the initial AMPsconcentration (affecting the peak concentration at the ITP interface)was examined on the binding reaction of AMPs to the bacteria membrane.FIG. 5A presents the labeling efficiency as a function of the appliedcurrent to the channel. The average labeling efficiency varies between65% and 85% for all the current values. The two sample t test method wasused to determine whether the difference between any two measured meanvalues is significant. Using a p-value of p=0.05, the calculatedt-values were significantly lower than the threshold requiredstatistical significance (for example, the calculated t-value for thedifference between the labeling efficiencies at 8 and 10 μA is 1.65,whereas the threshold based on the number of repeats is 2.78). Hence, toconclude, there is no significant advantage in the labeling efficiencyof one current over another. This is despite the fact that highercurrent results in higher electromigration velocity and, consequently,shorter reaction time. This indicates that, at these electric currentvalues, the labeling process is reaction limited and the advection timeof the bacteria through the labeling zone is significantly higher thanthe time required for binding. It is hypothesize that at sufficientlyhigh currents there would be a decrease in labeling efficiency as theadvection time decreases. However, the highest current presentedcorresponds to the maximum voltage (2200 V) possible in the experimentalsetup disclosed herein, and this decrease could not be experimentallyobserved. Within the range of currents tested, the highest current isthus optimal, as it provides the highest flow rate, without reducingefficiency. FIG. 5B presents experimental results of labeling efficiencyas a function of the AMPs concentration in the well, for a fixed currentof 3 μA. Consistent with theory, labeling efficiency increases with AMPsconcentration. It can concluded that the optimal concentration is 0.1μM, as beyond this value significant precipitation of AMPs was observed,with no significant gain in signal (applying a t test analysis, thecalculated t-value between the labeling efficiency at 0.1 and 1 μM is1.12, whereas the critical t-value for 95% confidence is 2.31),suggesting no significant difference in labeling efficiency.

The invention claimed is:
 1. A system comprising: (A) a protein having adomain that binds a bacterial membranal component; (B) a first flowchannel configured to provide flow of a liquid sample to anisotachophoresis (ITP) apparatus, said liquid sample comprising orsuspected of comprising a bacterial cell, a bacterial cell membrane or afraction of a bacterial cell membrane; and (C) wherein said ITPapparatus comprises (a) a first zone and a second zone, said first zoneis configured to contain a solution of high effective mobility leadingelectrolyte (LE) ion, and said second zone is configured to contain asolution of low effective mobility trailing electrolyte (TE) ion, saidfirst zone and said second zone are configured to be operably connectedto at least one anode and at least one cathode; and (b) at least onesecond flow channel elongated between said first zone and second zone;(D) a flow regulator, said flow regulator is configured to generate aflow countering an electromigration of said protein in the first flowchannel so as to maintain said protein in a pre-determined zone.
 2. Thesystem of claim 1, wherein said membranal component is a protein.
 3. Thesystem of claim 1, wherein said liquid sample is continuously injectedinto said first flow channel.
 4. The system of claim 1, wherein saidmembranal component is a net charge of phospholipid head groups orcharge density of phospholipid head groups.
 5. The system of claim 1,wherein said protein is labeled.
 6. The system of claim 5, wherein saidprotein is labeled by at least one label selected from the groupconsisting of: fluorescently labeled, chemiluminescently labeled,radioactively labeled, and colorimetrically labeled.
 7. The system ofclaim 1, wherein said protein is an anti-microbial peptide (AMP).
 8. Thesystem of claim 1, wherein said protein is an antibody or an antibodyfragment.
 9. The system of claim 1, comprising an anionic or cationicITP focusing stationary zone characterized by high concentration of saidprotein.
 10. The system of claim 1, wherein said LE comprises sodiumhydroxide and/or wherein said TE comprises Pyridine.
 11. The system ofclaim 1, further comprising a collector configured for collectinglabeled cells from said second zone.
 12. The system of claim 1, furthercomprising a detector selected from the group consisting of: aphotodetector, a photomultiplier tube (PMT), a conductivity detector, aradioactive detector, a camera and any combination thereof.
 13. Thesystem of claim 1, wherein said bacterial cell is an intact bacterialcell.
 14. A method for detecting a bacterial cell of interest, themethod comprising the steps of: a) providing a labeled protein having adomain that binds a membranal component in said bacterial cell ofinterest, wherein said labeled protein is focused by ITP to an ITP zonein a liquid flow channel; b) applying a counter flow so as to maintainsaid labeled protein in a stationary zone; c) providing a sample to saidflow channel, said sample comprising or suspected of comprising thebacterial cell of interest, a bacterial cell membrane or a fraction of abacterial cell membrane thereof; and d) detecting said bacterial cell ofinterest bound to said labeled protein.
 15. The method of claim 14,wherein said detecting includes labeling, separating, enriching,isolating, or any combination thereof.
 16. The method of claim 14,wherein said detecting is achieved by using a photodetector, aphotomultiplier tube (PMT), a conductivity detector, a radioactivedetector, a camera or any combination thereof.
 17. The method of claim14, wherein said labeled is: chemiluminescently labeled, radioactivelylabeled, or colorimetrically labeled.
 18. The method of claim 14,wherein said labeled protein is an anti-microbial peptide (AMP).
 19. Themethod of claim 14, wherein said labeled protein is an antibody.